Doerrier 2018 EBEC2018
Mitochondria (mt), the powerhouses of the cell, play a key role under many physio-pathological conditions. Therefore, the study of mitochondrial function is crucial for understanding such conditions, particularly in clinical applications. However, despite of the growing relevance of mitochondrial research, there is no database which provides comprehensive information about mitochondrial physiology. As a first step towards closing this gap, we developed the substrate-uncoupler-inhibitor titration (SUIT) reference protocol (RP) . RP comprises two complementary and harmonized SUIT protocols (RP1 and RP2) to investigate mt-pathways converging at the Q-junction from fatty acid oxidation (FAO or F), NADH-linked substrates (N), succinate (S) and alpha-glycerophosphate (Gp), allowing the identification of specific metabolic profiles and mt-related injuries. Here, we explain key points for SUIT protocol development and application of high-resolution respirometry (HRR) with a wide spectrum of mitochondrial preparations from different model organisms, tissues and cells. We highlight the use of a low malate concentration (0.1 mM) as a co-substrate with fatty acid(s), avoiding a significant overestimation of FAO encountered at 2 mM malate if anaplerotic pathways are involved and saturate the N-pathway with 2 mM malate only. An application of the SUIT-RP reveals defects in the N- and F-pathways and loss of mt-outer membrane integrity as a result of ischemia-reperfusion injury of mouse heart mitochondria. Respiratory OXPHOS analysis by HRR combining the evaluation of mitochondrial respiration with additional bioenergetic parameters (i.e., hydrogen peroxide production) provides a sensitive tool for comprehensive analysis of mitochondrial function in health and disease. Taken together, reproducibility, accuracy, application of comparable SUIT protocols and standardized normalization are required for functional mt-mapping as for a mt-database of mitochondrial physiology .
Labels: MiParea: Respiration, Comparative MiP;environmental MiP
Stress:Cryopreservation, Ischemia-reperfusion Organism: Human, Mouse Tissue;cell: Heart, Nervous system, Blood cells, HEK, Platelet Preparation: Permeabilized cells, Homogenate, Isolated mitochondria
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, Other combinations, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-FluoRespirometer Event: Poster MitoFit, MitoEAGLE
- Doerrier D(1), Sumbalova Z(1,2), Krumschnabel G(1), Garcia-Souza LF(1,3), Gnaiger E(1,4)
- Oroboros Instruments, Innsbruck, Austria - firstname.lastname@example.org
- Pharmacobiochem Lab, Fac Med, Comenius Univ, Bratislava, Slovakia
- Inst Sport Science, Univ Innsbruck, Austria
- Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria.
- Contribution to K-Regio project K-Regio MitoFit and European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.
- MitoEAGLE preprint 2018-06-10(39) Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1. - MitoEAGLE_preprint_2018-02-08.